In the preparation of multivalent conjugate pneumococcal vaccines directed to the prevention of invasive diseases caused by the organism Streptococcus pneumoniae (also known as pneumococcus), selected Streptococcus pneumoniae serotypes are grown to supply polysaccharides needed to produce the vaccine. The cells are grown in large fermentors with lysis induced at the end of the fermentation by addition of sodium deoxycholate (DOC) or an alternate lysing agent. The lysate broth is then harvested for downstream purification and the recovery of the capsular polysaccharide which surrounds the bacterial cells. After conjugation with a carrier protein, the polysaccharide is included in the final vaccine product and confers immunity in the vaccine's target population to the selected Streptococcus pneumoniae serotypes.
Although the cellular lysate produced in this process contains the target polysaccharide, it also contains large quantities of cellular debris including DNA, RNA, proteins, and residual media components. Traditional processing has involved a minimal pH reduction of the lysate to 6.6 by addition of acetic acid to help precipitate out the lysing agent and some of the impurities. This material is subjected to centrifugation followed by filtration to remove most of the solids down to a 0.45 μm nominal size. However, such traditional processing methods have shown minimal reduction in impurities with subsequent difficulty in removing soluble proteins to meet purified polysaccharide specifications.
The high burden of contaminating soluble protein has been particularly problematic within runs for certain serotypes. Some serotypes, in particular Streptococcus pneumoniae Type 3, produce large and viscous polysaccharide chains (e.g., for Type 3, chains of glucose/glucuronic acid of 2-3 million Daltons) that are released into the growth medium upon cellular lysis. Its viscosity has made it difficult to filter after centrifugation, and, in such cases, protein removal through the purification process has been insufficient and has led to run failures.
Accordingly, improved methods for the removal of protein impurities from complex cellular Streptococcus pneumoniae lysates, in particular lysates comprising Streptococcus pneumoniae Type 3 polysaccharides, are needed.